During the cooling and warming of tissues or cells in cryoprotective solutions, the cells and tissues are often injured. Adding and removing the cryoprotectant can also induce damage, particularly if the addition and washout process is prolonged but also if dilution is too rapid. It would be desirable in the art to provide a method that allows a) rapid addition of cryoprotectant, b) rapid or slow cooling without cold shock or chilling injury, and c) rapid dilution of the cryoprotectant without osmotic shock after rewarming of the living system.
There are many ways for living systems to be injured by cooling to subzero temperatures and subsequently warming from subzero temperatures. If the system is frozen, it may be damaged mechanically from intracellular or extracellular ice, or by consequences of changes in the composition of the unfrozen portion of the semi-frozen environment. If the system is vitrified, these sources of injury may disappear, but other forms of injury are possible.
“Thermal shock” (also sometimes called cold shock) is injury caused by rapid cooling per se, and is reduced by reducing the cooling rate. Thermal shock injury has been stated to exist (see Fahy et al., in Cell Biology of Trauma, J. J. Lemasters and C. Oliver, Eds, CRC Press, 1995, pp. 333-356) within a narrow temperature window between 0° C. and about −20° C. in rabbit renal cortex, but not below, but is currently believed not to be a major issue, even though rapid cooling rates are desirable for vitrification to reduce the likelihood of ice formation and cryoprotectant toxicity.
Another possible source of injury is “chilling injury,” which is a poorly-understood form of damage caused by exposure to low temperatures per se. Chilling injury is, for practical purposes, essentially not cooling rate dependent (except for small systems, which may escape from chilling injury at very rapid cooling rates), but instead is dependent primarily on the absolute temperature to which the system is cooled. Stated in another way, thermal shock is caused by rapid cooling and is reduced or eliminated by slow cooling, and chilling injury is caused by slow cooling and is not eliminated by rapid cooling, except perhaps possibly at very high cooling rates.
Another form of injury pertinent to vitrification (cryopreservation without ice formation on cooling) is devitrification. As the temperature in a vitrifiable system is brought close to the glass transition temperature, nucleation can generally occur. When the system is warmed up, nuclei formed in the vicinity of the glass transition temperature (Tg), which are unable to grow while the system remains below Tg, can grow and produce injury both intracellularly and extracellularly.
Another way of referring to injury that takes place upon cooling per se and that is not formally characterized as representing either thermal shock or chilling injury is to simply term such injury as being “cooling injury.”
The primary barriers to cryopreservation by vitrification are cryoprotectant toxicity, chilling injury, and devitrification. Cryoprotectant toxicity has been greatly reduced by recent inventions involving the use of weak glass-forming agents and qv* analysis and is no longer a major limiting factor for cellular systems. Because devitrification can be reduced using low-toxicity cryoprotectants, ice blockers, and rapid warming, it is also currently of diminishing concern. Chilling injury, however, remains a substantial and largely intractable problem which is difficult to address in part because of its unknown origin. Although there have been studies on the biochemistry of chilling injury in natural systems, nothing is known about the sources of chilling injury in cells that do not suffer from chilling injury at 0° C. and that have been loaded with vitrifiable concentrations of cryoprotectants and cooled to subzero temperatures. Therefore, it would be particularly valuable to be able to circumvent cooling injury in order to improve the results of vitrification.
One patented method is known in the art for dealing with chilling injury in such systems (see U.S. Pat. Nos. 5,962,214 and 5,723,282). Fahy showed that kidneys or kidney slices exposed to high but sub-vitrifiable concentrations of cryoprotectant at 0° C. were severely damaged by cooling to −20 to −30° C. He found that by using lower concentrations of permeating cryoprotectants prior to cooling, the sensitivity to chilling injury could be eliminated at these temperatures. Additional cryoprotectant could then be introduced by diffusion or by perfusion at −20 to −25° C., with reduced or eliminated toxicity (see also Fahy et al., in Cell Biology of Trauma, J. J. Lemasters and C. Oliver, Eds, CRC Press, 1995, pp. 333-356, but especially FIG. 8, pg. 353, and discussion thereof; and Khirabadi et al., Cryobiology 31: 597, 1994, and Cryobiology 32: 543-544, 1995.)
Surprisingly, this prior art method was later found to have a fatal flaw: when tissues protected at about −25° C. using the method were subsequently cooled to Tg, they actually suffered dramatically more severe injury than if they had been cooled directly from 0° C. (Khirabadi et al., Cryobiology 37: 447, 1998). Lack of permeation of the extra cryoprotectant at −25° C. was ruled out as a cause of the cooling injury, since doubling the equilibration time at −25° C. had minimal if any effect on cooling injury although it greatly exacerbated toxic injury at −25° C. Therefore, whatever its virtues may have been, this prior art method is untenable.
Fahy et al. explained the basis of their prior art method as follows (see the Cell Biology of Trauma reference above, pp. 351-352). “Human erythrocytes do not normally experience cold shock when cooled to 0° C., but are rendered susceptible to cold shock by exposure to hypertonic solutions prior to cooling . . . If the same hypertonicity is introduced at a temperature below 10° C., the cells remain uninjured either at the temperature of exposure or upon subsequent cooling. The method for avoiding cold shock, then, is to cool through the critical temperature range in the presence of low concentrations [low tonicity] and to increase concentration [tonicity] only after this critical temperature interval has been safely traversed.”
“In the case of the rabbit kidney, we were aware that osmotic shrinkage of tubular cells is induced by exposure to VS4 and V52, and that cell shrinkage appears to be required for cold shock injury in red cells. Since kidneys cooled to −30° C. in VS4 supported life about 60% of the time and kidneys cooled to −30° C. in V52 supported life 0% of the time, it seemed that a concentration not much below the 49% w/v concentration of VS4 should avoid cold shock entirely.”
In discussing the results of an experiment designed to test the analogy between thermal shock injury in red blood cells and cooling injury in rabbit kidney slices, Fahy et al. conclude (from the same reference): “It thus appears that the analogy between cooling injury in rabbit kidney tissue and cold shock in human erythrocytes is valid.”
Thus, it is clear that although much attention has been given to the problem of obviating cooling injury in the past, a method for preventing such injury is still needed.